Method for determining ion channel activity of a substance

ABSTRACT

A method for determining the ion channel activity of a substance comprises the steps of (i) expressing the substance as a heterologous protein in a host cell, and (ii) determining changes in permeability of the plasma membrane of the host cell induced by expression of the heterologous protein. A screening method for determining ion channel modulating activity of a test substance is also disclosed.

FIELD OF THE INVENTION

This invention relates to a method for determining ion channel activityof substances such as peptides, polypeptides and proteins and to amethod for screening potential therapeutic substances for their abilityto modulate ion channel function.

BACKGROUND OF THE INVENTION

Biological cells are encapsulated in a membrane made of a double layerof lipids separating the intracellular contents from the outside. Thelipid bilayer “sandwich” has a hydrophobic interior that preventsmovement of charged particles such as ions across it. However, there areprotein macromolecules that penetrate the membrane and act as portholesto allow ions to pass between the inside and outside of a cell. Thesestructures that allow rapid movements of ions (many millions per second)across a cell membrane, with no need for an immediate energy input, arecalled “ion channels”. The forces that influence the movement of ionsthrough a channel are electrical and chemical. The electrical force isthe electrical potential across the membrane, the chemical force is thedifference in concentration of an ion on the two sides of the membrane:the combination of the two is the electrochemical gradient for an ion.If the electrochemical gradient for an ion is not zero, ions will flowthrough a channel when it opens (as long as the channel lets themthrough).

There are many varieties of ion channels that differ in theirselectivity, methods of gating, conductance and kinetic properties.Channels can be selective for sodium ions, or for potassium ions, or forcalcium ions, or for chloride ions, or for protons etc and areclassified according to the ions that pass through them most freely. Forexample, sodium channels are more permeable to sodium than to any othercations or anions. Channels are also classified according to the way inwhich they are turned on or gated. For example, voltage-activatedchannels open or close in response to changes in membrane potential.Ligand-gated channels are turned on when ligands such asneurotransmitters or hormones bind to their surface. Proteins to whichligands bind are commonly called receptors and many receptors are partof the same macromolecule that forms the ion channel. However, somechannels are indirectly linked to receptors by second messenger systemsand the channel is then separate from the receptor. Channels can alsohave very different conductances. Conductance, the reciprocal ofresistance, is a measure of the ease with which ions pass through achannel and is given by the ratio of the current to the driving force.The conductance of different channels can range from picosiemens tohundreds of picosiemens (corresponding to resistances of 10⁹ to 10¹²ohms). Finally, channels can have very different “duty cycles”. Some areopen most of the time while others open infrequently. Some flickerrapidly between open and closed states while others do not. Changes inthe environment of channels (e.g the presence of drugs) can change thesecharacteristics. Indeed it is becoming clear that many drugs exert theireffects on cells and organs by binding to surface receptors andinfluencing channel behaviour.

The function of all cells in an animal or other organism depends on theion channels formed by membrane proteins which provide a pathway formovement of ions between compartments in a cell and between the interiorand exterior of cells. These movements of ions are essential for normalcell function, and all biological cells (including bacteria and evenenveloped viruses such as the influenza and HIV viruses) contain ionchannels. Ion channels are fundamental to cellular functions such astransmission of signals in nervous systems, cell division, production ofantibodies by lymphocytes, replication of virus particles within cellsand secretion of fluid and electrolytes.

A wide variety of diseases such as cystic fibrosis, musculardystrophies, stroke, epilepsy and cardiac arrhythmias are related todisorders of ion channel function. In addition, it has recently beendiscovered that some viruses have proteins that form ion channels thatare needed in the normal life-cycle of the virus. For example, there isnow good evidence that a protein (M2) in influenza A virus forms an ionchannel that is necessary for virus replication, and drugs such asamantadine that block this channel inhibit replication of the influenzaA virus. Amantadine (1-aminoadamantane hydrochloride) and its analoguerimantidine have been found empirically to be effective in theprophylaxis and treatment of influenza caused by the influenza A virus.These drugs, at the therapeutic concentrations, inhibit replication ofthe influenza A virus both in vitro and in vivo. However, they canbecome ineffective because of the development of resistant strains ofthe virus and this reduces their value as therapeutic agents.

Other drugs which work by modulating ion channel function includecalcium channel blockers which are used as anti-anginal andantihypertensive agents, barbiturates which cause sleep and inhibitepileptic seizures by increasing movements of chloride ions acrossreceptors activated by gamma-amininobutyric acid (GABA), andbenzodiazepines which relieve anxiety and produce anaesthesia byincreasing GABA receptor activity.

In the past, the discovery of drugs which block ion channels has beenlargely serendipitous. Drugs that have been discovered in this wayinclude general anaesthetics such as ether and halothane, thebarbiturates and benzodiazepines. Thus, ether was originally used likealcohol at parties, and the reversible anaesthetic effect of halothanewas discovered during leakage of refrigerant from a compressor.Similarly, the discovery of the antiarryhthmic action of quinidinefollowed use of quinine as an antimalarial drug.

Realisation that ion channels could prove to be an important site ofdrug action has lead to a search for effective ways of screening theactivity of potential therapeutic substances that affect ion channelactivity. Although electrophysiological techniques can be used to detectcurrent flow when ions move across channels, the methods are too tediousand time-consuming for routine screening of ion channel activity.

Vpu is a small phosphorylated integral membrane protein encoded by theHIV-1 genome which associates with the Golgi and endoplasmic reticulummembranes in infected cells, but has not been detected in the plasmamembrane nor in the viral envelope. The protein is 80-82 amino acidslong depending on the viral isolate, with an N-terminal transmembraneanchor and a hydrophilic cytoplasmic C-terminal domain. The C-terminaldomain contains a 12 amino-acid sequence that is conserved in allisolates and contains two serine residues that are phosphorylated. Usingstandard techniques associated with reconstitution of the purified HIV-1Vpu protein in planar lipid bilayers, it has been shown that the Vpuprotein forms cation selective ion channels in phospholipid bilayers(8). Further work is now directed to finding drugs that block thesechannels, and testing them as potential anti-HIV-1 therapeutic agents.While screening for such drugs is possible using the above mentionedplanar lipid bilayer method, this method has the disadvantage ofrequiring large quantities of highly purified Vpu protein and is limitedin that only one compound can be tested per bilayer, making it arelatively slow and inefficient screening assay.

Because of these disadvantages, there is a need for an ion channel assaysystem that can be used both to detect the ion channel activity ofbiologically important peptides and proteins, and to screen theeffectiveness of potential therapeutic substances that might interactwith ion channels and modulate ion channel function.

Some organisms such as bacteria accumulate amino acids and othersubstances by using the energy of a cation concentration gradient. If asubstance such as a peptide, polypeptide or protein that forms a channelis inserted in the cell membrane and dissipates the gradient, theorganism can no longer accumulate essential substances and growth isinhibited. This growth inhibition can be detected directly. Thus, theactivity of potential therapeutic substances that might influence thefunction of a channel can be quickly screened by examining their effectson growth of an organism containing the channel-forming peptide,polypeptide or protein.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for determiningion channel activity of a substance which is a peptide, polypeptide,protein or the like, which comprises the steps of:

(i) expressing said substance as a heterologous protein in a host cell;and

(ii) determining changes in permeability of the plasma membrane of saidhost cell induced by expression of said heterologous protein.

Preferably, the determination of changes in the permeability of theplasma membrane of the host cell is carried out by detecting changes inthe permeability of the plasma membrane to small metabolite molecules,for example proline or adenine.

In accordance with this aspect of the invention, if the test substanceis expressed as a heterologous protein having ion channel activity,expression of the heterologous protein in the plasma membrane of thehost cell will alter the ability of the cell to maintain concentrationgradients of small metabolite molecules such as proline or adenine whosetransport into the cell is energised by the ions which are permeable tothe expressed channel. As a result, a net movement or leakage of themetabolite molecules out of the cell will occur, and such leakage of themetabolite molecules can then be detected by a suitable method.Preferred methods for detecting leakage of the metabolite molecules fromthe cell are described below.

In a further aspect, the present invention provides a screening methodfor determining ion channel modulating activity of a test substance,which comprises the steps of:

(i) expressing a substance having ion channel activity as a heterologousprotein in a host cell;

(ii) contacting said host cell with the test substance; and

(iii) determining changes in ion channel activity of said heterologousprotein induced by the test substance.

Preferably, in this aspect of the invention, changes in ion channelactivity of the heterologous protein induced by the test substance aredetermined by detecting the effect of the test substance on changes inpermeability of the plasma membrane of the host cell induced byexpression of the heterologous protein in the cell; in particular, bydetecting the effect of the test substance on changes in thepermeability of the plasma membrane of the host cell to small metabolitemolecules such as proline, adenine or the like.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment, the assay method of the present inventionmeasures the alteration of the permeability to small metabolitemolecules (proline or adenine, for example) of the plasma membrane ofliving host cells (E. coli, for example) induced by the expression ofheterologous cation (sodium, for example) channel proteins (Vpu, forexample) in the host cells. Although the following detailed descriptionis directed specifically to Vpu ion channels, it will be understood thatthe concept of the assay is generally applicable to any ion channelprotein that can be actively expressed in a host cell such as E. coli.

The plasma membrane of a cell generally contains proteins whose functionis the uptake of metabolite molecules into the cell. In a subset ofthese proteins, the energy to drive the uptake reaction is derived fromtransmembrane concentration gradients of various ions (eg Na+, H+) suchthat the movement across the membrane and into the cell of themetabolite to be taken up is tightly coupled to the movement across themembrane of an ion moving down its concentration gradient. If aheterologous channel forming protein is present in the membrane of thecells and causes the dissipation of the concentration gradient of theion driving the uptake of a metabolite, then a net movement of themetabolite out of the cell should occur—particularly in the case wherethe metabolite can be derived biosynthetically by the cell. Leakage ofthe metabolite from cells expressing the ion channel can be detected,for example : (i) by either the ability of the leaked metabolite tosupport the growth of a second type of cell that has an auxotrophicrequirement of the leaked metabolite; or (ii) in the case wherebiosynthesis of the metabolite is rate limiting to growth, by thefailure of cells expressing the heterologous channel forming protein tothrive in the absence of externally supplied metabolite.

As a specific example of the first detection method, the E. coli prolinetransporter is driven by the co-transport into the cell of sodium ionswith proline. Cross-feeding between a strain of E. coli expressing theHIV-1 Vpu protein—which consequently leaks proline due to dissipation ofthe sodium gradient—and a second strain of E. coli that cannotsynthesise proline but instead must take it up from the external mediumhas been demonstrated. Such experiments are performed in prolinedeficient medium so that the only possible source of proline is viabiosynthesis in the Vpu-expressing strain.

As a specific example of the second detection method, the expression ofVpu in E. coli strain XL-1 Blue at 37° C. makes cell growth dependent onexternally supplied adenine. The same strain in the absence of Vpuexpression grows well when adenine is absent from the growth media.

The in vivo assay of ion channel function described above also has theadvantages of speed and efficiency over the planar lipid bilayer assay(8) as a method for screening potential therapeutic substances thatmight block, inhibit or otherwise modulate the ion channel function asmany (hundreds) such substances can be screened in a single experiment.Thus the present invention also provides a method for rapidly screeningcompounds for their ability to block, inhibit or otherwise modulate thefunction of ion channel proteins expressed in living cells.

As previously described, the assay method relies on expression of theion channel forming proteins in the plasma membrane of the cells,altering the ability of the cell to maintain concentration gradients ofthe metabolites whose transport into the cells is energised by the ionswhich are permeable to the expressed channel. Leakage of the metabolitefrom the cell is preferably detected by one of two methods:

(i) cross-feeding of a second strain of cells which are auxotrophic forthe leaked metabolite; or

(ii) failure to thrive of the cells expressing the ion channel in theabsence of the leaking metabolite supplied in the external medium.

Preferably, the expression system involves the expression of ion channelproteins in E. coli from their corresponding genes (preferably cDNAsegments) cloned into E. coli plasmid expression vectors. Such vectorconstruction and expression in E. coli uses the standard methodsassociated with E. coli genetics and molecular biology, described by wayof example, by Sambrook et al.(9).

One preferred embodiment of the method of the present invention arisesfrom the observation of cross-feeding between two cell lines—preferablybacterial cells—induced in response to ion channel activity of theexpressed foreign gene(s). In the specific case where E. coli cells arebeing used and a sodium channel is being expressed (for example asdetailed further below), the leakage of proline (a metabolite whosetransport into cells is energised by the sodium gradient) from thechannel-expressing cells can be detected by cross-feeding of a secondstrain of E. coli that is auxotrophic for proline (i.e. unable tosynthesise proline). Control experiments to establish that the expressedchannel is not inducing a non-specific leak of all small moleculesthrough the cell membrane would be set up identically to detectmethionine leakage. The E. coli methionine transporter is energised byATP hydrolysis and therefore the absence of a sodium gradient should notinduce leakage of methionine out of the cells.

As described above, the present invention also extends to a method forscreening potential therapeutic substances that may act as ion channelinhibitors. This screening method is a simple extension of the assaymethod described above, which in one preferred embodiment involvessetting up the cross-feeding assay in the same way as previouslydescribed, with the addition of the various substances to be tested tothe cells expressing the ion channel protein. Substances which block orinhibit the ion channel activity would prevent dissipation of thepermanent ion gradient, and would thereby not induce leakage ofmetabolites. Control experiments could be performed simultaneously toensure the substances being tested do not affect the normal growth of E.coli. If such substances are found, they would be excluded fromscreening by the cross-feeding assay.

Further features of the present invention are more fully described inthe following Example(s). It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention, and should not be understood in any way as arestriction on the broad description of the invention as set out above.

In the accompanying drawings:

FIGS. 1A-1C Plasmids used for the expression of Vpu in E. coli. A. Theamino acid sequence encoded by the Vpu open reading frame (ORF)generated by PCR from an HIV-1 strain HXB2 cDNA clone (SEQ ID No.1), asdescribed in Example 1. The Vpu ORF was cloned in-frame at the 3′ end ofthe GST gene in p2GEX to generate p2GEXVpu (B). It was subsequentlycloned into pPL451 to produce the plasmid pPL-Vpu (C).

FIGS. 2A-2B Expression and purification of Vpu in E. coli. A. Westernblotting after SDS-PAGE was used to detect expressed Vpu in E. coliextracts. Lanes 1-4 contain samples, at various stages of purity, of Vpuexpressed from p2GEXVpu: lane 1, GST-Vpu fusion protein isolated byglutathione-agarose affinity chromatography; lane 2, Vpu liberated fromthe fusion protein by treatment with thrombin; lane 3, Vpu purified byHPLC anion exchange chromatography; lane 4, Vpu after passage throughthe immunoaffinity column. Lanes 5 and 6, membrane vesicles preparedfrom 42° C. induced cells containing pPL-Vpu or pPL451, respectively. B.Silver stained SDS-PAGE gel: lane 1, Vpu purified by HPLC anion exchangechromatography; lane 2, Vpu after passage through the immunoaffinitycolumn.

FIGS. 3A-3C Bacterial cross-feeding assays. A full description of thisassay is given in Example 1. For all plates, the Met-, Pro- auxotrophicstrain was used to seed a soft agar overlay. Plates A and B containminimal medium supplemented with methionine; in plate C the medium wassupplemented with proline. To control for viability of the cells in thebackground lawn, the discs labelled P and M contained added proline ormethionine, respectively. The discs labelled C and V were inoculatedwith Met+, Pro+ E. coli cells containing the plasmids pPL451 or pPL-Vpu,respectively. Plates were incubated at 37° C. (A and C) or 30° C.(B) fortwo days and photographed above a black background with peripheralillumination from a fluorescent light located below the plate. Theimages were recorded on a Novaline video gel documentation system. Lighthalos around the discs labelled P or M on all plates and around the disclabelled V on plate A indicate growth of the background lawn strain.

FIGS. 4A-4D NADH-dependent Atebrin fluorescence quenching from evertedplasma membrane vesicles prepared from E. coli cells expressing Vpu (B)or the influenza B protein NB (C). Control vesicles were prepared fromstrains containing the appropriate expression vectors (A and D). NADHaddition and the time at which the cuvette solution goes anaerobic areindicated by the arrows.

FIGS. 5A-5B The adenine growth-dependency assay. Expression of Vpu fromthe plasmic pPL-Vpu at 38° C. makes growth of the host E.coli cellsdependent on the presence of adenine in the external media. Panel A:Minimal media agarose plate with E. coli cells containing pPL-Vpustreaked on the left hand side and cells containing pPL-451 (no Vpugene) on the right hand side. Panel B: An identical plate and cellstreaks, except that adenine (0.002% wt/v final) was added to the mediabefore the plate was poured. Note that the Vpu expressing cells growwell when adenine is present in the plate.

FIGS. 6A-6B Use of the adenine growth-dependence assay to detect mutantVpu ion channels with altered channel activity. Panel A representssections from a minimal medium agar plate (containing no adenine) ontowhich XL-1 Blue E.coli cells have been streaked containing either: 1.,pPL-Vpu (expressing wild-type Vpu); 2., pPL-VpuRK37DI (expressing amutant form of Vpu); or 3., pPL-451 (control plasmic with no Vpu gene).The plate was then incubated at 38° C. for two days. While the cellsexpressing wild-type Vpu did not grow (apart from a few revertantcolonies), those expressing the Vpu mutant can clearly be seen to begrowing, albeit not as efficiently as the no Vpu control cells.

When the RK37DI mutant Vpu protein was tested in the bilayer assay(Panel B) the channels were found to have a conductance of 3picosiemens, which is approximately 20% of the wild-type channelconductance. The residual activity might explain why the cellscontaining the mutant plasmid did not grow as well as the no-Vpu controlcells.

FIGS. 7A-7B Use of the adenine growth-dependence assay to screen fordrugs which inhibit Vpu ion channel activity. Panel A represents asection from a minimal medium agar plate (containing no adenine) on towhich a lawn of XL-1 Blue E. coli cells expressing wild-type Vpu(pPL-Vpu) has been seeded. At the numbered circles, 1 μl of a solutionof various drugs has been applied to the plate and allowed to soak intothe agar. The plate was then incubated at 38° C. for two days. At #'s1-4 the drug has had no effect on Vpu's ability to prevent cell growth.At #5 a solution containing an excess of adenine was applied as acontrol —the bright ring around #5 indicates growing E.coli cells. At#6, compound ANU-9 was added—the faint but detectable ring of growthindicated a potential inhibition of Vpu channel activity by ANU-9. Thiswas confirmed when ANU-9 was tested in the bilayer assay: Panel B showspartial inhibition of Vpu channel activity at 50 μM ANU-9 and completeinhibition at 250 μM.

EXAMPLE 1

This example demonstrates the functional expression of Vpu in E. colihost cells and the detection of changes in the permeability of theplasma-membrane of the host cells to proline by detecting leakage ofproline from the host cells using the cross-feeding method. It will beunderstood that the same methods can be performed to demonstrate ordetermine the ion channel activity of peptides, polypeptides or proteinsother than Vpu.

Construction of recombinant plasmids p2GEXVpu and pPL-Vpu

The open reading frame encoding Vpu (SEQ ID No. 1—FIG. 1A) was amplifiedby PCR from a cDNA clone of an Nde1 fragment of the HIV-1 genome(isolate HXB2, a gift from Dr N. Deacon, McFarlane Burnet Centre,Melbourne, Australia). Native Pfu DNA polymerase (Stratagene; 0.035U/μl) was chosen to catalyse the PCR reaction to minimise possible PCRintroduced errors by virtue of the enzyme's proofreading activity. The5′, sense, primer (AGTAGGATCCATGCACCTATACC—SEQ ID No.2) introduces aBamH1 site (underlined) for cloning in-frame with the 3′ end of the GSTgene in p2GEX (1). This primer also repairs the start codon (bold Treplaces a C) of the vpu gene which is a threonine codon in the HXB2isolate. The 3′, antisense, primer (TCTGGAATTCTACAGATCATCAAC—SEQ ID No.3) introduces an EcoR1 site (underlined) to the other end of the PCRproduct to facilitate cloning. After 30 cycles of 94° C. for 45 sec, 55°C. for 1 min and 72° C. for 1 min in 0.5 ml thin-walled eppendorf tubesin a Perkin-Elmer thermocycler, the 268 bp fragment was purified,digested with BamH1 and EcoR1 and ligated to p2GEX prepared by digestionwith the same two enzymes. The resultant recombinant plasmid p2GEXVpu,is illustrated in FIG. 1B. The entire Vpu open reading frame and theBamH1 and EcoR1 ligation sites were sequenced by cycle sequencing, usingthe Applied Biosystems dye-terminator kit, to confirm the DNA sequence.

To prepare the Vpu open reading frame for insertion into the pPL451expression plasmid (2), p2GEXVpu was first digested with BamH1 and the5′ base overhang was filled in with Klenow DNA polymerase in thepresence of dNTPs. The Vpu-encoding fragment was then liberated bydigestion with EcoR1, purified from an agarose gel and ligated intopPL451 which had been digested with Hpa1 and EcoR1. Western blotssubsequently confirmed that the pPL-Vpu construct (FIG. 1C) expressedVpu after induction of cultures at 42° C. to inactivate the cI857represser of the PR and PL promoters.

The pPL-Vpu construct was then inserted into E. coli host cells usingknown techniques (9).

Growth and expression characteristics of pPL-Vpu

On agar plates made from rich medium (e.g. Luria Broth supplemented withglucose), E. coli cells containing pPL-Vpu grew when incubated at 30° C.and 37° C. but not at 42° C., while control strains grew well at 42° C.Liquid cultures of cells containing pPL-Vpu were grown at 30° C. toOD600=0.84 then moved to grow at 42° C. for two hours (the final celldensity was OD600=0.75) The plasma membrane fraction was prepared andwestern blotting detected a single band at approximately 16 kDa,indicating that Vpu was expressed and associated with the membranes(FIG. 2A, lane 5).

Cross-feeding experiments reveal that proline leaks out of cellsexpressing Vpu

Uptake of proline by E. coli is well characterised and active transportof the amino acid into the cells is known to use the sodium gradient asthe energy source (3). It was predicted that if the sodium gradient weredissipated by a sodium channel in the plasma membrane then prolinesynthesised in the cytoplasm will diffuse out of the cells. To detectwhether this proline leakage occurred, the following cross-feeding assaywas used: A lawn of an E. coli strain auxotrophic for proline andmethionine (Met− Pro−), was seeded and poured as a soft agar overlay onminimal media plates lacking proline but containing methionine. Sterileporous filter discs were inoculated with a Met+ Pro+ strain (XL-1 blue)containing either the pPL451 control plasmid or pPL-Vpu and placed ontothe soft agar. The plates were then incubated at 37° C. or 30° C. fortwo days. After that time a halo of growth of the Met- Pro- strain wasclearly visible surrounding the disc inoculated with the cellscontaining pPL-Vpu incubated at 37° C. (FIG. 3A). This growth can onlybe due to the leakage of proline from the Vpu-expressing cells on thedisc. No such leakage was apparent from the control strain at 37° C. noraround either strain on plates grown at 30° C. (FIG. 3B).

Methionine does not leak out of cells expressing Vpu

In contrast to proline transport, the E. coli methionine permease isknown to belong to the ABC transporter family (4) and hence be energisedby ATP. Identical cross-feeding experiments to those described abovewere set up except that the Met− Pro− strain was spread on minimalplates lacking methionine but containing proline. No growth of thisstrain was evident around any of the discs (FIG. 3C), indicating thatmethionine was not leaking out of the XL-1 blue cells even when Vpu wasbeing expressed.

Proton permeability of membrane vesicles is unaffected by the presenceof Vpu

To investigate whether the Vpu sodium-conductive channel expressed in E.coli membranes was also permeable to H+, the NADH-dependent atebrinfluorescence quenching assay (5) was used. This technique can be used tomeasure the ability of E. coli membrane vesicles to maintain a protongradient generated by the electron transport chain during oxidativephosphorylation. The fluorescent atebrin molecule contains twoprotonatable nitrogen atoms. The unprotonated form is electricallyneutral and is able to equilibrate between the interior and exterior ofthe vesicles. The increased internal concentration of protons, generatedin the presence of NADH ADP and oxygen, results in protonation ofatebrin molecules that are inside the vesicles and the subsequent netaccumulation of atebrin inside the vesicles results in quenching of itsfluorescence. Vesicles leaky to protons, and hence unable to maintain ahigh H+in/H+out ratio, do not quench atebrin fluorescence as efficientlyas control vesicles.

In this study, membrane vesicles prepared from E. coli cells expressingVpu from pPL-Vpu were not more proton permeable than control vesiclesprepared from the background strain (FIGS. 4, A and B). The Vpu proteinwas present in the membranes (see FIG. 2A, lanes 5 & 6) and it cantherefore be concluded that it had not formed a channel permeable enoughto protons to be detected by the fluorescence quenching technique.

The NB protein of influenza B has been shown to form cation-selectivechannels in bilayers (6) and may be equivalent to M2 of influenza Awhich has been shown to be a hydrogen ion channel (7). Membrane vesicleswere prepared from a strain containing the plasmid pQE+NB. Thesevesicles contained the NB protein by western analysis (not shown) andhad clearly reduced atebrin fluorescence quenching activity compared tothe control strain (FIG. 4C and D), confirming that the NB channels arepermeable to hydrogen ions. The fluorescence quenching technique isclearly capable of detecting the presence of proton-Vpu conductingchannels and this control experiment provides support for the conclusionthat the protein does not form a proton-conducting channel whenexpressed in E. coli.

EXAMPLE 2

This example demonstrates the functional expression of Vpu in E. colicells and the detection of changes in the permeability of the plasmamembrane of the host cell to adenine by detecting failure to thrive ofthe host cells when grown on minimal medium plates lacking adenine. Aswith Example 1, it will be understood that the same methods can beperformed to demonstrate or determine the ion channel activity ofpeptides, polypeptides or proteins other than Vpu.

The same expression and control plasmids are used as described inExample 1 above (pPL-Vpu and pPL451, respectively). When cells of the E.coli strain XL-1 Blue containing the Vpu expression plasmid pPLVpu areincubated at 37° C. on minimal medium plates the host cells fail to grow(FIG. 5). Because of an undefined temperature sensitive mutation in theadenine biosynthesis pathway of E. coli strain XL-1 Blue, this strain isunable to up-regulate adenine biosynthesis in response to adenineleakage induced as a result of Vpu sodium channel expression. Growth ofthese Vpu-expressing cells can be restored if adenine is included in thenutrient medium at sufficiently high concentration to negate the netdriving force for loss of this molecule from the cells.

In contrast, the same host cells containing the control plasmid pPL451(which is identical to pPL-Vpu except for the absence of the DNA segmentencoding the Vpu protein), grow normally at 37° C. on minimal mediumplates in the absence (or presence) of adenine (FIG. 5).

These observations indicate that expression of Vpu in the XL-1 Bluecells has caused leakage of adenine from the cells in a manner analogousto the proline leakage described in Example 1. In this case, the testfor a functioning sodium channel expressed in the E. coli plasmamembrane is the inability of the host cells to grow in the absence ofadenine.

EXAMPLE 3

This example demonstrates the use of the adenine growth-dependence assayto detect mutant forms of the Vpu protein affecting channel activity.

A site directed mutation was introduced to the Vpu gene so as to changethe amino acids Arg and Lys as positions 37 and 38 to Glu and Ile in theprotein expressed from the mutated gene (called “RK37DI mutant Vpu”). Inelectrophysiological assay of channel function this mutation was shownto reduce the conductance of the ion channels formed to approx 20% ofthat of the wild-type channels (3 picosiemens versus 15 picosiemens,respectively). FIG. 6B shows a comparison of the size of the currentsproduced by mutant and wild-type channels in planar lipid bilayers.

On minimal media plates (containing no adenine—as per FIG. 5), cellscontaining the plasmid encoding the RK37DI mutant Vpu had a partialgrowth phenotype compared to cells containing the wild-type Vpu gene(which don't grow at all) and to cells containing no Vpu gene (in whichgrowth is unaffected)—See FIG. 6.

This result illustrates the correlation between the biological (adeninegrowth dependence) assay and the in-vitro (electrophysiological) assayin terms of their abilities to reflect Vpu channel activities.

EXAMPLE 4

This example demonstrates the screening method of the present inventionfor screening test substances for ion channel inhibitory propertiesusing the methods of Examples 1 and 2 to obtain functional expression ofVpu in E. coli host cells and detecting leakage of proline or adeninefrom the host cells using the cross-feeding method.

For cases in which the cross-feeding method is being employed to detectthe channel activity (as in Example 1), filter discs inoculated with thechannel-expressing host cells are subjected to addition of small volumesof solution containing the substance(s) to be tested. If the added druginhibits channel activity, then cross-feeding of the background strainis not observed.

For cases in which adenine requirement for growth is being employed todetect the channel activity (as in Example 2), the XL-1 Blue host cellsexpressing the channel protein are spread to form a lawn of cells onminimal medium plates lacking adenine. The test substance(s) is thenapplied to defined areas of the plates and growth of the XL-1 Blue cellsaround the area in which the test substance(s) is applied indicateschannel inhibition has occurred to prevent adenine leakage (FIG. 7A).

As an example, a compound (ANU-9) has been identified which, when addedat a discrete location to such a minimal medium plate (no adenine) asdescribed above, allows E. coli cells expressing Vpu to grow in a regionsurrounding the point of application (FIG. 7A). This indicates that inthe region of growth compound ANU-9 is at a concentration sufficient toinhibit the Vpu ion channel such that cell growth can occur in theabsence of adenine.

ANU-9 was subsequently screened in the electrophysiological assay forits ability to block Vpu ion channel activity (FIG. 7B). At 50 μMchannels were severely inhibited, with only infrequent, small openingsdetected (middle trace FIG. 7B), while at 250 μM channel activity wascompletely inhibited (lower trace FIG. 7B).

Detection of ion channel modulating activity of a test substance

A lawn of an E. coli strain auxotrophic for proline and methionine (Met−Pro−), is seeded and poured as a soft agar overlay on minimal mediaplates lacking proline but containing methionine. Sterile porous filterdiscs are impregnated with a test substance to be screened andinoculated with a Met+ Pro+ strain (XL-1 blue) containing the pPL-Vpuconstruct (prepared as described in Example 1) and placed onto the softagar. The plates are then incubated at 37° C. or 30° C. for two days.After that time a halo of growth of the Met− Pro− strain is clearlyvisible surrounding the disc inoculated with the cells containingpPL-Vpu if the test substance is one that does not block the Vpu ionchannel. If the test substance is one that does block the Vpu ionchannel, no growth of the Met− Pro− strain is observed around the disc.A control experiment is performed whereby a disc impregnated with thetest substance is used to show that the test substance has no effect onthe normal growth of E. coli.

Persons skilled in this art will appreciate that variations andmodifications may be made to the invention as broadly described herein,other than those specifically described without departing from thespirit and scope of the invention. It is to be understood that thisinvention extends to include all such variations and modifications.

REFERENCES

1. Piller S c, Ewart G D, Premkumar A, Cox G B, and Gage P W, (1996),Vpr protein of human immunodeficiency virus type 1 formscation-selective channels in planar lipid bilayers, Proceedings of theNational Academy of Sciences of the United States of America 93:111-115.

2. Love C A, Lilley P E, and Dixon N E, (1996), Stable high-copy numberbacteriophage lambda promoter vectors for overproducton of proteins inEscherichia coli. Gene. 176:49-53.

3. Yamato I, Kotani M, Oka Y, and Anraku Y, (1994), Site-speicificalteration of arginine 376, the unique positively charged amino acidresidue in the mid-membrane-spanning reginos of the proline carrier ofEscherichia coli. Journal of Biological Chemistry, 269:5729-5724.

4. Rosen B R, ATP-coupled solute transport systems, in Escherichia coliand Salmonella typhimurium: Cellular and molecular biology, F. C.Neidhardt, Editor. 1987, American Society for Microbiology: WashingtonD.C., p. 760-767.

5. Haddock B A and Downie J A, (1974), The reconstitution of functionalrespiratory chains in membranes from electron-transport-deficientmutants of Escherichia coli as demonstrates by quenching of atebrinfluorescene. Biochem. J. 142:703-706.

6. Sunstrom N A, Premkumar L S, Premkumar A, Ewart G and Cox G B,(1996), Ion channels formed by N B, and influenze B virus protein.Journal of Membrane Biology 150:127-132.

7. Schroeder C, Ford C M, Wharton S A, and Hay A J, (1994), Functionalreconstitution in lipid vesicles of influenza virus M2 protein expressedby baculovirus: evidence for proton transfer activity, J. Gen Virol.75:3477-3484.

8. Ewart G D, Sutherland T, Gage P W, and Cox G B, (1996). The Vpuprotein of HIV-1 forms cation selective ion channels. J. Virol.70:7108-7115.

9. Sambrook J, Fritsch E F, and Maniatis T, (1989). Molecular Cloning: ALaboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

3 1 82 PRT HIV-1 1 Met Gln Pro Ile Pro Ile Val Ala Ile Val Ala Leu ValVal Ala Ile 1 5 10 15 Ile Ile Ala Ile Val Val Trp Ser Ile Val Ile IleGlu Tyr Arg Lys 20 25 30 Ile Leu Arg Gln Arg Lys Ile Asp Arg Leu Ile AspArg Leu Ile Glu 35 40 45 Arg Ala Glu Asp Ser Gly Asn Glu Ser Glu Gly GluIle Ser Ala Leu 50 55 60 Val Glu Met Gly Val Glu Met Gly His His Ala ProTrp Asp Val Asp 65 70 75 80 Asp Leu 2 24 DNA Artificial SequenceDescription of Artificial Sequence5′ sense primer 2 agtaggatccatgcaaccta tacc 24 3 24 DNA Artificial Sequence Description ofArtificial Sequence3′ antisense primer 3 tctggaattc tacagatcat caac 24

We claim:
 1. A screening method for determining ion channel modulatingactivity of a test substance having potential for such modulatingactivity, which comprises the steps of: (i) expressing a peptide,polypeptide or protein in the plasma membrane of a host cell, saidpeptide, polypeptide or protein having ion channel activity whenexpressed as a heterologous protein in the plasma membrane of the hostcell; (ii) contacting said host cell with the test substance; and (iii)determining changes to the ion channel activity of said heterologousprotein induced by the test substance, wherein the changes to the ionchannel activity of the heterologous protein induced by the testsubstance are determined by detecting the effect of the test substanceon changes in net movement across the plasma membrane of the host cellof small cellular metabolite molecules which do not directly permeatethe ion channel formed by said heterologous protein.
 2. A methodaccording to claim 1, wherein the effect of the test substance onchanges in the net movement of proline or adenine molecules across theplasma membrane is detected.
 3. A method according to claim 1, whereinsaid host cell is E. coli.
 4. A method according to claim 1, whereinsaid substance having ion channel activity is a heterologous cationchannel protein.
 5. A method according to claim 4, wherein saidsubstance having ion channel activity is a heterologous sodium channelprotein.
 6. A method according to claim 4, wherein said substance havingion channel activity is the HIV-1 Vpu integral membrane protein.
 7. Amethod according to claim 1, wherein leakage of said small cellularmetabolite molecules from the host cell is detected.
 8. A methodaccording to claim 7, wherein leakage of said small cellular metabolitemolecules from the host cells is detected by either: (i) cross-feedingof cells which are auxotrophic for the leaked metabolite; or (ii)failure of cells expressing the ion channel to grow in the absence ofthe leaking metabolite being supplied in the external medium.
 9. Amethod for determining ion channel modulating activity of a testsubstance having potential for such modulating activity, which comprisesthe steps of: (i) expressing a peptide, polypeptide or protein in theplasma membrane of a host cell, said peptide, polypeptide or proteinhaving ion channel activity when expressed as a heterologous protein inthe plasma membrane of the host cell; (ii) contacting said host cellwith the test substance; and (iii) determining changes to the ionchannel activity of said heterologous protein induced by the testsubstance, wherein the changes to the ion channel activity of theheterologous protein induced by the test substance are determined bydetecting the effect of the test substance on changes in permeability ofthe plasma membrane of the host cell to small cellular metabolitemolecules, wherein said heterologous protein having ion channel activityis the HIV-1 Vpu integral membrane protein.
 10. The method of claim 9wherein the effect of the test substance on changes in the permeabilityof the plasma membrane to proline or adenine molecules is detected. 11.The method of claim 9 wherein said host cell is E. coli.
 12. The methodof claim 9 wherein leakage of metabolite from the host cell is detected.13. The method of claim 12 wherein leakage of metabolite from the hostcells is detected by either: (i) cross-feeding of cells which areauxotrophic for the leaked metabolite; or (ii) failure of cellsexpressing the ion channel to grow in the absence of the leakingmetabolite being supplied in the external medium.
 14. A screening methodfor determining ion channel modulating activity of a test substancehaving potential for such modulating activity, which comprises the stepsof: (i) expressing HIV-1 Vpu integral membrane protein in the plasmamembrane of a host cell, said protein having ion channel activity whenexpressed as a heterologous protein in the plasma membrane of the hostcell; (ii) contacting said host cell with the test substance; and (iii)determining changes to the ion channel activity of said heterologousprotein induced by the test substance, wherein the changes to the ionchannel activity of the heterologous protein induced by the testsubstance are determined by detecting the effect of the test substanceon changes in net movement across the plasma membrane of the host cellof small cellular metabolite molecules.
 15. The method of claim 14wherein the effect of the test substance on changes in the movement ofproline or adenine molecules is detected.
 16. The method of claim 14wherein said host cell is E. coli.
 17. The method of claim 14 whereinleakage of metabolite from the host cell is detected.
 18. The method ofclaim 17 wherein leakage of metabolite from the host cells is detectedby either: (i) cross-feeding of cells which are auxotrophic for theleaked metabolite; or (ii) failure of cells expressing the ion channelto grow in the absence of the leaking metabolite being supplied in theexternal medium.
 19. A screening method for determining ion channelmodulating activity of a test substance having potential for suchmodulating activity, which comprises the steps of: (i) expressing HIV-1Vpu integral membrane protein in the plasma membrane of a host cell,said protein having ion channel activity when expressed as aheterologous protein in the plasma membrane of the host cell; (ii)contacting said host cell with the test substance; and (iii) determiningchanges to the ion channel activity of said heterologous protein inducedby the test substance.
 20. The method of claim 19 wherein the effect ofthe test substance on changes in the permeability of the plasma membraneto proline or adenine molecules is detected.
 21. The method of claim 19wherein said host cell is E. coli.
 22. The method of claim 19 whereinleakage of metabolite from the host cell is detected.
 23. The method ofclaim 22 wherein leakage of metabolite from the host cells is detectedby either: (i) cross-feeding of cells which are auxotrophic for theleaked metabolite; or (ii) failure of cells expressing the ion channelto grow in the absence of the leaking metabolite being supplied in theexternal medium.